Project summary The proposed work is part of our long-term goal to understand host-transposon interactions that influence eukaryotic genome function and evolution. Retrotransposons are a class of transposable element capable of amplifying their copy number via reverse-transcription of RNA intermediates and insertion in a new locus. Through this transposition activity, they can multiply to the point of making up the majority of genetic material in some genomes, exerting a broad influence in genome structure and regulation. The repetitive nature of TE can also affect genome integrity through homologous recombination between their dispersed copies that can cause chromosomal rearrangements. To counteract their deleterious effects, eukaryotes have evolved multiple genome defense mechanisms that suppress Retrotransposon activity. The most important are RNA interference (RNAi) pathways that are often specifically active in the germline, protecting the genetic material that will make up the next generation. We are investigating these host-Retrotransposon interactions using the LTR Retrotransposons Tf1 and Tf2, endemic to fission yeast. In previous work, we have revealed an intense cross-regulation between the Retrotransposon and the process of host DNA replication, discovering how Retrotransposons select new insertion sites, regulate chromatin silencing, and control homologous recombination. Several aspects of these phenomena are conserved in LTR retrotransposons present in the genomes of other organisms, even while the specific factors involved are not. This suggests that common evolutionary pressures lead to convergent evolution of host-Retrotransposon interactions. As a consequence, these conserved mechanisms may be important for metazoan germline stability. In the present application, we propose a comprehensive line of research combining genetics, biochemistry, and high throughput sequence-based genomics methods to (1) ascertain universal Retrotransposon insertion site selection mechanisms, (2) resolve the determinants of LTR Retrotransposon detection by RNAi, (3) determine the mechanism by which Retrotransposons guide their own homologous recombination, and (4) investigate the effect of Retrotransposon activity in meiosis progression. In pursuit of these goals, we will make use of novel fission yeast strains in which all Retrotransposon copies have been deleted through an innovative CRISPR mutagenesis method. This advance enables us to carry out previously unfeasible genetic analyses of these highly repetitive elements. The proposed studies will provide a novel and comprehensive understanding of host- Retrotransposon interactions, and their consequences on genome regulation and stability.